The timing of cell division is coordinated with other cyclic events, of other periodicities, in the lives of cells. From bacteria, to algae, to diverse cll types in mammals, the circadian biological clock controls the time of day during which cell division can occur. The mechanism and function of this time restriction, or gating, of cell divisio is poorly understood in any system, and such regulatory checkpoints in mammals are important factors in developmental programs and the progression of cancer. The overall goal of this project is to understand how and why circadian rhythms and cell division are interlocked. The circadian control of cell division in the cyanobacterium Synechococcus elongatus provides an elegant system in which to address questions that probe the interactions of the clock, the cytokinesis machinery, and the segregating chromosomes. This project seeks to answer: What is the biological role of the circadian checkpoint of cytokinesis? What are the components of the machinery that connect the circadian clock to cell division? Where are the clock oscillator components during the cell and circadian cycles and how are they inherited? And, is the partitioning of chromosomes in S. elongatus, whose ploidy levels oscillate with circadian rhythmicity, related to the gating of cell division? The specific aims will: (1) Define the components, through which the circadian clock regulates cell division, and determine the consequences of bypassing the cell division gate; (2) Discern the intracellular localization and dynamics of clock proteins during the circadian and cell division cycles; and (3) Elucidate the relationships among the clock, cytokinesis, and chromosome segregation. Existing mutants that bypass the circadian cell division checkpoint will be used to test hypotheses that address the role of the gate in protecting circadian precision, chromosome integrity, and/or cell-to-cell variations in gene expression. Mass spectrometry will identify factors that associate with the cell division machinery during the gating checkpoint. Time-lapse imaging of cells trapped in microfluidics chambers will enable simultaneous monitoring of cell division and circadian rhythms to assess the consequences of gating in individual wild-type cells and in mutants that bypass the circadian gate. Super-resolution Structured Illumination imaging will provide sufficient sensitivity and resolution to track the localization and dynamics of circadian oscillato proteins and tagged chromosomes in wild-type and mutant cells throughout the circadian and cell division cycles. Sorting and imaging flow cytometry methods will be used to assess ploidy changes in wild-type and mutant strains. This project will reveal how and why a circadian clock controls cell division, a coupling of timing circuits that occurs in mammalian cells as well as in cyanobacteria, and will provide novel insight into how cells inherit a sense of time. PUBLIC HEALTH RELEVANCE: This project will reveal how and why cell division is controlled by the circadian clock, a phenomenon that occurs in mammalian cells as well as in cyanobacteria. It will also provide insight into the faithful distribution of chromosomes to cells and the control of chromosome copy number, which in humans are factors in developmental programs and the progression of cancer.